Increased mass levels of certain serine proteases in the stratum corneum in acute eczematous atopic skin.

Acute eczematous atopic dermatitis (AD) is associated with increases in stratum corneum (SC) serine protease activity. The purpose of this study was to examine whether the increased SC protease activities in acute eczematous atopic dermatitis were associated with increased mass levels of SC proteases. Six subjects with healthy skin and six patients with AD each with non-lesional skin or lesional acute eczematous skin had the mass levels of their extractable SC kallikreins (KLK), plasmin and urokinase quantified using Luminex multiplex bead-based assays from SC tape strippings. The mass levels of KLK5 and KLK14 together with urokinase were not elevated in the SC in atopic skin. However, the mass levels of KLK7 and KLK11 together with plasmin were greatly elevated compared with the extracts from the non-lesional and the healthy skin and correlated with the corresponding enzymatic activities.

Immunopharmacological in vitro effects of Eleutherococcus senticosus extracts.

The objective of these investigations was to further elucidate the immunopharmacological profile of fluid extracts of Eleutherococcus senticosus and to identify the specific role of its characteristic eleutherosides B and E. An ethanolic dry extract of Eleutherococcus senticosus was used as starting material for the isolation of the eleutherosides B and E. Immunopharmacological studies included expression of major histocompatibility complex class I and II molecules by rat bone marrow-derived mononuclear phagocytes, human lymphocyte marker flow cytometry, and in vitro testing of human lymphocyte functions. In contrast to the isolated eleutherosides B and eleutherosides E and the re-mixed eleutherosides B and E, the whole ethanolic fluid extract of Eleutherococcus senticosus was able to induce and enhance interleukin-1 and interleukin-6 but not interleukin-2 production in vitro. The effective concentration of the whole ethanolic extract ranged from 1.0-0.1 mg/ml for the enhancement of interleuking-1 alpha production and 1.0-0.03 mg/ml for the enhancement of interleukin-6 production. It is concluded that the observed enhancing immunopharmacological activities on acute phase response mediators are best exhibited by the induction with whole ethanolic extracts whereas the species-specific and characteristic eleutherosides B and E are not associated with these activities.

Mode of action of plasmolysed yeast on lymphocytes under microgravity stress.

A newly developed device to simulate microgravity for space biological investigations under laboratory conditions allowed us to apply a reproducible environmental stress on immunologically active cells. Cell proliferation, soluble IL-2 receptor in the culture supernatant, lymphocyte surface activation markers like CD25 (IL-2R), CD69 and HLA-Dr were the endpoints measured. Untreated donor lymphocyte reactions under microgravity were compared to the same cells treated with an immunomodulator from herbal plasmolysed yeast (Bio-Strath Food Supplement). The main finding is the enhancement of the proliferation inhibition under microgravitational stress by the herbal plasmolysed yeast.

Stress-compensation by a food supplement based on yeast plasmolysate in mitogen-activated T lymphocytes under simulated low-gravity.

T lymphocyte function is strongly depressed in vitro and in vivo under low-g conditions in space as well as simulated in clinostat. Here we describe the effect of a food supplement based on yeast plasmolysate on T cells activated in vitro with Concanavalin A and cultured in a random positioning machine. The mitotic index was measured by 3H-thymidine incorporation into DNA, the expression of activation markers CD25, CD69 and HLA-DR on the cell surface by cytofluorimetry and the secretion of the IL-2R by an enzyme immunoassay. Our data indicate that the food supplement used is capable to modulate T lymphocyte function. The addition of the food supplement increased the expression of activation markers in activated and non-activated cells. Cultivation under low-gravity conditions reduced the expression of the activation markers, but this expression was partly restored or even increased upon addition of yeast plasmolysate. On the other hand, cell proliferation and secretion of soluble IL-2 receptor was reduced after addition of the food supplement in all samples.

Stimulation of cytokine production via a special standardized mistletoe preparation in an in vitro human skin bioassay.

The mistletoe preparation Lektinol is standardized with respect to bioactive mistletoe lectin, the active component of mistletoe. This standardized mistletoe preparation and its active components (mistletoe lectins) were compared in the skin2 bioassay in vitro for their capacity to stimulate interleukin 1 alpha and interleukin 6 release from skin analogue tissue, composed of human cells in their naturally secreted matrix. The standardized mistletoe preparation, its basic ingredient, aqueous mistletoe extract, and pure mistletoe lectins all stimulated IL-1 alpha and IL-6 release from skin2 tissues during 24 h incubation. The amounts of cytokines released from various skin2 tissue lots by mistletoe lectin I (ML I) (0.75-8.0 ng/ml) and by the standardized mistletoe preparation remained relatively constant across a series of different batches. Concentration-response curves to the standardized mistletoe preparation and ML I were similar for IL-1 alpha and IL-6 release. The importance of the concentration of mistletoe lectins for the cytokine-releasing action of the standardized mistletoe preparation was confirmed using a neutralizing anti-mistletoe lectin antiserum. CONCLUSIONS. Using the skin2 method it was shown that reproducible stimulation of cytokine release by a standardized mistletoe preparation from batch to batch is one of the notable features of its pharmaceutical quality. This standardized mistletoe preparation therefore represents a preparation with constant immunobiological effects. Mistletoe lectins of the standardized mistletoe preparation are the active substances in the skin2 bioassay. The skin2 method is a reliable quantitative bioassay for determination of immunopharmacological effects.

Macrophage response to microbial pathogens: modulation of the expression of adhesion, CD14, and MHC class II molecules by viruses, bacteria, protozoa and fungi.

The ability of inactivated viruses, bacteria, protozoa and fungi to modulate the expression of CD14, CD49d, CD49f, CD11a (LFA-1), and CD54 (ICAM-1) molecules in unprimed bone marrow-derived mononuclear phagocytes (BMM phi) was investigated by means of flow cytometry. Incubation with bacterial agents resulted in the large majority of experimental situations in enhanced expression of these macrophage surface molecules. In contrast, viruses and fungi down-regulated the expression of several adhesion receptors, especially integrins. Amplification of MHC class II expression triggered in macrophages by interferon gamma was clearly inhibited by viruses, bacteria, protozoa and fungi. The findings explain earlier results showing that, under the same experimental conditions, bacterial agents are, for the most part, potent stimulators of secretory and cell-mediated macrophage activities while viruses, protozoa and fungi are poor in this respect.

Coordinate up- and down-modulation of inducible nitric oxide synthase, nitric oxide production, and tumoricidal activity in rat bone-marrow-derived mononuclear phagocytes by lipopolysaccharide and gram-negative bacteria.

Simultaneous incubation of primary rat bone-marrow-derived mononuclear phagocytes (BMMo) and tumor cells with gram-negative agents triggers within 24 h interferon gamma (IFN gamma)- and tumor necrosis factor (TNF alpha)-independent tumoricidal activity. On the other hand, BMMo that had been incubated for 24 h with gram-negative agents prior to re-exposure to the same agent had largely lost their ability to generate tumoricidal activity, although their ability to bind lipopolysaccharide (LPS) was not diminished. Parallel measurements of the kinetics of inducible nitric oxide synthase (iNOS), nitrite secretion, and tumoricidal activity triggered in primary BMMo by LPS revealed that these parameters take a coordinate course, reaching a peak within 24 h and then rapidly decaying. Down-regulation of expression of NOS protein and iNOS activity could be attributed neither to down-regulation of LPS receptors nor to L-arginine depletion.

Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target.

Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AMø). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AMø from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AMø consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AMø do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.

[Functional IgG subclass determinations in patients with suspected humoral immune defects using the LIBA technique]. / Funktionelle IgG-Subklassenuntersuchungen bei Patienten mit Verdacht auf humoralen Immundefekt mittels LIBA-Technik.

In patients with recurrent infections, "intrinsic" asthma and lymphoma the specific binding capacity of IgG subclass 2 against pneumococcus was determined by line-immuno-binding-assay (LIBA). The results show that LIBA represents a simple and specific test system suitable as diagnostic parameter in patients with a suspected humoral defect.

Macrophage response to bacteria and bacterial products: modulation of Fc gamma receptors and secretory and cellular activities.

The ability of bacteria and bacterial products to modulate the expression of Fc gamma receptors and major histocompatibility complex (MHC) class II molecules in resting rat bone marrow-derived mononuclear phagocytes (BMM phi) was determined by means of flow cytometry (FCM). Binding of IgG via Fc gamma receptors was considerably enhanced by most microbial agents; bacterial lipopolysaccharide, lipoteichoic acid and some intact bacteria proved to be as active as interferon-gamma (IFN-gamma) and augmented binding of IgG via high- and low-affinity Fc gamma receptors. In contrast, expression of MHC class II molecules by BMM phi was only slightly affected by the microbial agents. Additional findings attest that resting unprimed rat BMM phi are able to respond directly to Gram-negative and Gram-positive bacteria and to some of their products with the expression of marked secretory [in particular tumour necrosis factor-alpha (TNF-alpha) and nitrite] and cellular activities (TNF-alpha-independent tumoricidal activity). This extensive, direct type of macrophage activation may substantially amplify the capability of these cells to cope with these infectious agents in first-line, non-specific host defence.

Mitogenic signal transduction in T lymphocytes in microgravity.

The activation by concanavalin A Con A of human peripheral blood lymphocytes (PBLs) in the presence of monocytes as accessory cells was investigated in cultures exposed to microgravity conditions in Spacelab. Activation of T cells was measured as incorporation of [3H]thymidine into DNA, secretion of interleukin-2 (IL-2), and interferon-gamma, and expression of IL-2 receptors. Whereas, as discovered in earlier experiments, the activation of resuspended T cells is strongly inhibited, activation of cells attached to microcarrier beads is more than doubled in microgravity. The results suggest that the depression of the activation in resuspended cells may be attributed to a malfunction of monocytes acting as accessory cells. In fact, although the ultrastructure of resuspended monocytes is not altered in microgravity, the secretion of IL-1 is strongly inhibited. Our data suggest that (1) IL-2 is produced independently of IL-1, (2) IL-1 production is triggered only when monocytes (and lymphocytes?) adhere to microcarriers, (3) the expression of IL-2 receptors depends on IL-1, and (4) provided sufficient IL-1 is available, activation is enhanced in microgravity. Finally, cultures of resuspended PBLs and monocytes in microgravity constitute a complete and natural system in which monocytes are not operational. This may be useful for studies of the role of accessory cells and cell-cell interactions in T lymphocyte activation.

Mononuclear phagocytes from human bone marrow progenitor cells; morphology, surface phenotype, and functional properties of resting and activated cells.

After 3-4 weeks culture of human bone marrow cells in medium supplemented with IL-3, macrophage- (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), the firmly adherent cells exhibited the morphologic features of mononuclear phagocytes and were strongly esterase-positive. Flow cytometric analysis revealed a rather homogeneous cell population with marked autofluorescence; the large majority of the cells expressed CD14, CD11a, b, and c, Fc receptors for IgG, Fc gamma RI, II, and III, and HLA class II molecules. Interferon-gamma (IFN-gamma), bacteria, and bacterial products modulated expression of some of the surface markers, induced and/or enhanced respiratory burst, phagocytic activity, secretion of tumour necrosis factor, and tumouricidal activity; in contrast, these cells were not able to generate reactive nitrogen intermediates.

Effect of head-down tilt bedrest (10 days) on lymphocyte reactivity.

Immunological responses of six healthy males to 10 days of head-down tilt bedrest (HDT) were assessed. Lymphocyte responsiveness was severely reduced immediately before, during, and immediately after the HDT, even though the lymphocyte numbers did not change. By contrast, delayed-type hypersensitivity was not affected. No dramatic changes were found in WBC counts and lymphocyte subpopulations, with the only exception of natural killer (NK) cells which transiently decreased immediately after HDT. Plasma cortisol levels were elevated above normal immediately before and during the HDT. The data suggest that the mitogenic response of lymphocytes was affected by psychological and fluid shift stress. These results are compared with data obtained during and after spaceflight. We conclude that the stress of HDT induces changes in immunological responsiveness that are strikingly similar to those arising from the stress of spaceflight.

Binding of monomeric immunoglobulins by bone marrow-derived mononuclear phagocytes; its modulation by interferon-gamma.

The ability of resting and activated rat bone marrow-derived mononuclear phagocytes (BMM phi) to bind monomeric rat, mouse, and human IgG was determined by means of flow cytometry. Rat IgG2b bound with high affinity (Kd approximately equal to 3 x 10(-9) M); binding was optimal at 4 degrees C and was only little affected by trypsin treatment. The other IgG bound with only low affinity (rat IgG2a, mouse and human IgG) or not at all to rat BMM phi (rat IgG1, rat IgG2c). The binding of rat IgG2b was not affected by the presence of a surplus of low-affinity binding IgG, and vice versa, indicating that high- and low-affinity IgG bind to different sites. Binding of high- and low-affinity IgG as well as expression of MHC class II molecules and of tumoricidal activity by BMM phi was markedly enhanced by rat interferon-gamma in low concentration (0.1 to 1.0 IU IFN-gamma/ml). On the other hand, heat-killed Corynebacterium parvum organisms, that were equally potent in triggering tumoricidal activity, neither enhanced the binding of IgG nor the expression of MHC class II molecules by BMM phi, suggesting that these abilities are not necessarily closely related phenomena.

Effect of a herbal yeast food supplement and long-distance running on immunological parameters.

The effect of a food supplement on immunological parameters of 16 long-distance runners was tested in a randomized, double-blind and placebo-controlled trial. The supplement comprised plasmolysed herbal yeast, malt, honey, and orange juice. No statistically significant differences between the two groups regarding the following variables were detected at three sessions at rest and immediately after a 21 km run: total and differential white blood cell counts, numbers of B- and T-cells and T-subpopulations, concanavalin-A-induced lymphocyte proliferation, serum levels of immunoglobulins, neopterin, IL-2 receptors, beta 2-microglobulin, complement factor b, c4 and c3c, and c1-inactivator. These findings suggest that the effects of the tested food supplement on these parameters are negligible with respect to improvements in the immunological status of long-distance runners. The changes observed immediately after the run had a transient character. In both groups, however, low lymphocyte counts, IgG subclass 2 levels and c1-inactivator levels were noted at rest, which indicate that the immune status of endurance athletes may be affected by training.

Repopulation of the atrophied thymus in diabetic rats by insulin-like growth factor I.

Atrophy of the thymus is one of the consequences of severe insulin deficiency. We describe here that the weight and the architecture of the thymus of diabetic rats is restored towards normal not only by insulin but also by insulin-like growth factor I (IGF-I) treatment. In contrast to insulin, this effect of IGF-I occurs despite persisting hyperglycemia and adrenal hyperplasia. We also investigated the in vivo effect of IGF-I on replication and differentiation of thymocytes from streptozotocin-induced diabetic rats. Thymocytes from diabetic rats incorporated less [3H]thymidine than did thymocytes from healthy rats. Insulin, as well as IGF-I treatment of diabetic rats increased [3H]thymidine incorporation by thymocytes. Flow cytometry of thymocytes labeled with monoclonal antibodies revealed a decreased expression of the Thy-1 antigen in diabetic rats compared with control rats. In addition, a major deficiency of thymocytes expressing simultaneously the W3/25 and the Ox8 antigens (corresponding to immature human CD4+/CD8+ thymocytes) was observed. These changes were restored towards normal by insulin as well as by IGF-I treatment. The antibody response to a T cell-dependent antigen (bovine serum albumin) was comparable in normal and diabetic rats. We conclude that IGF-I has important effects on the thymocyte number and the presence of CD4+/CD8+ immature cells in the thymus of diabetic rats despite persisting hyperglycemia. However, helper T-cell function for antibody production appears to be preserved even in the severely diabetic state.

Surface phenotype of rat bone marrow-derived mononuclear phagocytes.

Utilizing a panel of currently available monoclonal antibodies, the surface phenotype of a pure population of resting rat bone marrow-derived mononuclear phagocytes (BMM phi) was analyzed by means of flow cytometry. The present work provides an extensive list of surface markers expressed by BMM phi and also outlines advantages and limitations of flow cytometric analysis of this cell type. The results show that the majority of surface markers considered to be expressed selectively by T lymphocytes, such as Thy-1, CD2 and CD5 antigens, leukosialin (W3/13), or an alloantigen of peripheral T cells, are not expressed by BMM phi. On the other hand, the CD8 antigen and the leukocyte common antigen recognized by MRC OX-33, considered to represent specific markers of cytotoxic T cells and/or peripheral B cells, are expressed on a variable, often considerable proportion of BMM phi. Monoclonal antibodies W3/25, MRC OX-35, and MRC OX-38, directed against epitopes on the CD4 molecule, labeled a variable proportion of BMM phi. Among the 39 monoclonal antibodies examined, none appeared to recognize an epitope which is expressed selectively by mononuclear phagocytes.

Modulation of major histocompatibility complex (MHC) expression by interferons and microbial agents. Independent regulation of MHC class II expression and induction of tumoricidal activity in bone marrow-derived mononuclear phagocytes.

In a pure population of rat bone marrow-derived mononuclear phagocytes (BMM phi), the expression of major histocompatibility complex (MHC) molecules and ability to manifest tumoricidal activity were simultaneously studied. Resting BMM phi, which express low levels of MHC class II molecules and do not manifest tumoricidal activity, become strongly MHC class II-positive, and evolve tumoricidal activity within 24 h when incubated with macrophage-activating lymphokines (MAF) or gamma interferon (IFN-gamma). In contrast, BMM phi which were interacted for 24 h with heat-killed microbial agents (Corynebacterium parvum, Listeria) evolve tumoricidal activity without parallel enhancement of MHC class II expression. IFN-alpha,beta neither induced tumoricidal activity nor enhanced MHC class II expression. Further experiments have shown that (a) the kinetics of MAF- and/or IFN-gamma-induced amplification of MHC class II expression and of tumoricidal activity are different; (b) enhancement of MHC class II expression by rIFN-gamma is not invariably paralleled by induction of tumoricidal activity; and (c) inhibitors of macrophage tumoricidal activity differ in their ability to affect MHC class II expression. It is concluded from these findings that in a population of pure BMM phi, i.e. in the complete absence of lymphocytes, the expression of MHC molecules and induction of tumoricidal activity are independently regulated phenomena; in particular, the enhanced expression of MHC class II molecules is not a prerequisite for induction and/or manifestation of tumoricidal activity by mononuclear phagocytes.

Effect of long-term physical exercise on lymphocyte reactivity: similarity to spaceflight reactions.

The response of critical immunological parameters in seven athletes to the sustained physical stress of marathon running was assessed. Variables analysed were the responsiveness of lymphocytes (measured as mitogenic response to concanavalin A), the numbers of lymphocytes, their subsets, and leukocyte numbers. In addition, blood levels of cortisol, epinephrine, and norepinephrine were determined. After the run, lymphocyte responsiveness was severely depressed to 1-70% of the resting values, even though the lymphocyte counts did not change. Leukocyte counts were elevated 2.8-fold. No dramatic changes were found within the lymphocyte subsets, although an increase in pan T-cells and the helper/inducer subset 2 d after the run was significant. In addition, the numbers of B-cells decreased significantly. No change was observed within the suppressor/cytotoxic subset. Cortisol increased 2.1-fold, epinephrine 3.2-fold and norepinephrine 2.7-fold. All these parameters returned to baseline values within 2 d. These data were compared with data obtained during and after spaceflight. We conclude that prolonged physical stress of marathon running induces changes in immunological responsiveness that are strikingly similar to those arising from the stress of spaceflight.